| Autor: |
Figueiredo N; MARE-Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal., Matos B; MARE-Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal., Diniz M; UCIBIO-Applied Molecular Biosciences Unit, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal., Branco V; Research Institute for Medicines (iMed.ULisboa), Faculty of Pharmacy, Universidade de Lisboa, 1649-003 Lisboa, Portugal., Martins M; MARE-Marine and Environmental Sciences Centre, Departamento de Ciências e Engenharia do Ambiente, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal.; UCIBIO-Applied Molecular Biosciences Unit, NOVA School of Science and Technology (FCT NOVA), 2829-516 Almada, Portugal. |
| Abstrakt: |
Primary cell cultures from wild organisms have been gaining relevance in ecotoxicology as they are considered more sensitive than immortalized cell lines and retain the biochemical pathways found in vivo . In this study, the efficacy of two methods for primary hepatocyte cell isolation was compared using liver from two marine fish ( Sparus aurata and Psetta maxima ): (i) two-step collagenase perfusion and (ii) pancreatin digestion with modifications. Cell cultures were incubated in L-15 medium at 17 ± 1 °C and monitored for up to six days for cell viability and function using the trypan blue exclusion test, MTT test, lactate dehydrogenase (LDH) activity, and ethoxyresorufin O-deethylase (EROD) activity after Benzo[a]Pyrene exposure. The results showed significant differences between the number of viable cells ( p < 0.05), the highest number being obtained for the pancreatin digestion method (average = 4.5 ± 1.9 × 10 7 cells). Moreover, the hepatocytes showed solid adherence to the culture plate and the rounded shape, changing into a triangular/polygonal shape. The cell viability and function obtained by pancreatin digestion were maintained for five days, and the EROD induction after exposure to the B[a]P showed that cells were metabolically active. This study shows that the optimized pancreatin digestion method is a valid, cost-effective, and simple alternative to the standard perfusion method for the isolation of primary hepatocytes from fish and is suitable for ecotoxicological studies involving marine pollutants, such as PAHs. |
